Use of representations of DNA for genetic analysis

• US 20040137473A1
• Filed: 10/01/2003
• Published: 07/15/2004
• Est. Priority Date: 10/30/1997
• Status: Abandoned Application

• Current Assignee: Cold Spring Harbor Laboratory
• Original Assignee: Cold Spring Harbor Laboratory
• Inventors: Lucito, Robert, Wigler, Michael
• Application Number:
  US10/677,396
• Publication Number:
  US 20040137473A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

It is an object of the present invention to provide a solution to problems associated with the use of microarray technology for the analysis DNA. The present invention provides compositions and methods for the use of simple and compound representations of DNA in microarray technology. The present invention is also directed to methods for the production of High Complexity Representations (HCRs) of the DNA from cells.

Claims

1. An array of nucleic acid probes comprising probes derived from one or more representations of DNA.

2. The array of claim 1, wherein the DNA is genomic DNA.

3. The array of claim 1, wherein the DNA is from a megacloning vector.

4. The array of claim 1, wherein the DNA is from cDNAs.

5. The array of claim 3, wherein the megacloning vector specific sequences, and host specific sequences are substantially removed by representational difference analysis.

6. The array of claim 2, wherein the representation has a complexity equal to that of 70% or less of the complexity of genomic DNA.

7. The array of claim 2, wherein the representation has a complexity equal to that of 20% or less of the complexity of genomic DNA.

8. The array of claim 2, wherein the representation has a complexity equal to that of 5% or less of the complexity of genomic DNA.

9. A compound representation of DNA consisting essentially of those fragments of a first representation that do not possess a predetermined second restriction endonuclease site, said second restriction endonuclease site being different from that recognized by a first restriction endonuclease used to make the first representation.

10. A compound representation of DNA consisting essentially of portions of those fragments of a first representation that possess a second predetermined restriction endonuclease site, said second restriction endonuclease site being different from that recognized by a first restriction endonuclease used to make the first representation.

11. A microarray being hybridized to one or more representations of DNA.

12. The microarray of claim 11, wherein two representations are being hybridized to the microarray.

13. The microarray of claim 12, wherein the two representations are compound representations.

14. The microarray of claim 12, wherein the representations are differentially labeled.

15. A method of producing a compound representation of DNA, comprising:

(a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments;

(b) ligating an adaptor to said digested DNA fragments;

(c) amplifying said DNA fragments using primers complementary to said adaptors to provide a first representation of said DNA;

(d) digesting the first representation with a second restriction endonuclease to provide digested DNA fragments;

(e) amplifying said DNA fragments of step (d) using primers complementary to said adaptors to provide a compound representation of said DNA.

16. A method of producing a compound representation of DNA, comprising:

(a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments;

(b) ligating a first adaptor to said digested DNA fragments;

(c) amplifying said DNA fragments using primers complementary to said adaptors to provide a first representation of said DNA;

(d) digesting the first representation with a second restriction endonuclease to provide digested DNA fragments;

(e) ligating a second adaptor to the ends of said fragments of step (d) created by the second restriction endonuclease;

(f) ligating a third adaptor to the 5′

ends of said fragments of step (d);

(g) amplifying said DNA fragments of step (f) using primers complementary to the second and third adaptors to provide a compound representation of said DNA.

17. A method of hybridizing nucleic acids from one or more samples to an array of probe DNA immobilized on a surface of a solid phase comprising:

contacting said array, containing or suspected of containing sequences complementary to nucleic acids from one or more samples, with nucleic acids from one or more samples, under conditions such that hybridization between the nucleic acids and probe DNA can occur, wherein the one or more sample nucleic acids are or are derived from one or more representations.

31. Use of the method of claim 17 to detect variations in gene copy number, variations in levels of gene expression, loss of heterozygosity, genetic polymorphisms, or to determine paternity or to map a sample nucleotide to a particular member of a megacloning vector library.

18. A method of hybridizing nucleic acids from one or more samples to an array of probe DNA immobilized on a surface of a solid phase comprising:

contacting said array, containing or suspected of containing sequences complementary to the nucleic acids from one or more samples, with the nucleic acids from one or more samples, under conditions such that hybridization between the nucleic acids and probe DNA can occur, wherein the probe DNA is or is derived from one or more representations.

32. Use of the method of claim 18 to detect variations in gene copy number or variations in levels of gene expression.

19. A method of hybridizing nucleic acids from one or more samples to an array of probe DNA immobilized on a surface of a solid phase comprising:

contacting the array, containing or suspected of containing sequences complementary to the nucleic acids from one or more samples, with the nucleic acids from one or more samples, under conditions such that hybridization between the nucleic acids and probe DNA can occur, wherein the one or more sample nucleic acids are or are derived from representations and wherein the probe DNA is or is derived from one or more representations.

33. Use of a the method of claim 19 to detect variations in gene copy number, variations in levels of gene expression, loss of heterozygosity, genetic polymorphisms, or to determine paternity or to map a sample nucleotide to a particular member of a megacloning vector library.

20. A method of producing a high complexity representation of DNA, comprising:

(a) digesting a sample of DNA with a relatively frequent cutting restriction endonuclease to provide digested DNA fragments;

(b) ligating adaptors to said digested DNA fragments; and

(c) amplifying said DNA fragments using primers complementary to said adaptors to provide a high complexity representation of said DNA.

21. The method of claim 20 in which the sample of DNA is obtained from a biopsy specimen, a cell line, an autopsy specimen, a forensic specimen or a paleoentological specimen.

22. The method of claim 21 in which the biopsy specimen is a fractioned biopsy specimen or a microdissected biopsy specimen.

23. The method of claim 20 in which the sample of DNA is obtained from a fixed cell or tissue specimen.

24. The method of claim 20 in which the restriction endonuclease is selected from the group consisting of DpnII, Tsp509I, MboI, Sau3Al, MaeII, MspI, HpaII, BfaI, HinPI, Csp6l, TaqI, MseI, AluI, BstUI, DpnI, HaeIII, RsaI, HnaI, and NlaIII.

25. The method of claim 20 in which the amplifying step (c) entails amplification by the polymerase chain reaction.

26. The method of claim 20 in which the DNA sample is a genomic DNA sample and the high complexity representation represents about 20-90% of the genome.

30. Use of the high complexity representation obtainable according to the method of claim 20 or claim 27 for archiving a DNA sample.

27. A method of producing a high complexity representation of DNA, comprising:

(a) digesting a sample of DNA with at least two restriction endonucleases to provide digested DNA fragments in which at least about 20% of said fragments are at between 100 and 1000 kb;

(b) ligating adaptors to said digested DNA fragments; and

(c) amplifying said DNA fragments using primers complementary to said adaptors to provide a high complexity representation of said DNA.

28. The method of claim 27 in which the DNA sample is a genomic DNA sample and the high complexity representation represents about 20-90% of the genome.

29. Use of one or more high complexity representations for genetic analysis in a method selected from the group consisting of measuring sequence copy numbers by Southern blotting, measuring sequence copy number by quantitative PCR;

monitoring loss of heterozygosity and monitoring genomic alterations through hybridization to chromosome spreads or panels of DNA probes.

34. Use of one or more high complexity representations as a template for the polymerase chain reaction.

35. A method of hybridizing nucleic acids from one or more samples to an array of probe DNA immobilized on a surface of a solid phase comprising:

contacting said array, containing or suspected of containing oligonucleotides complementary to nucleic acids from one or more samples, with nucleic acids from one or more samples, under conditions such that hybridization between the nucleic acids and probe DNA can occur, wherein the probe DNA comprises oligonucleotides complementary to and derived from one or more representations.

36. The method of claim 35, further comprising the step of extending those oligonucleotides hybridized to said sample nucleic acids by incubating said array in the presence of polymerase and deoxynucleotides under conditions such that extension can occur.

37. The method of claim 35, further comprising the step of extending those oligonucleotides hybridized to said sample nucleic acids by incubating said array in the presence of polymerase and one or more dideoxynucleotides under conditions such that extension can occur.

38. The method of claim 37, wherein the one or more dideoxynucleotides are labeled dideoxynucleotides.